This application claims priority of German Patent Application No. 101 15 578.6, filed Mar. 29, 2001, incorporated by reference herein.
The invention relates to a method for compensating for imaging defects in scanning microscopy and to a scanning microscope.
In scanning microscopy, a specimen is illuminated by a light beam in order to observe the reflected or fluorescent light thereupon emitted by the specimen, laser beams usually being used for illumination. In this case, a specimen is scanned by means of a finely focused light beam. The focus of the illumination light beam is moved in a specimen plane with the aid of a controllable beam deflection device, which generally has two tiltable mirrors. In this case, the deflection axes are usually perpendicular to one another, so that one mirror deflects the incident beam in the x-direction and the other deflects the beam in the y-direction. The tilting of the mirrors is achieved for example with the aid of galvanometer actuating elements.
Especially in confocal scanning microscopy, a specimen is scanned with the focus of a light beam in three dimensions. A confocal scanning microscope generally comprises a light source, a focusing optic by which the light from the source is focused onto a pinhole aperturexe2x80x94the so-called excitation aperturexe2x80x94a beam splitter, a beam deflection device for beam control, a microscope optic, a detection aperture and the detectors for registering the detection or fluorescent light. The illumination light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back via the beam deflection device to the beam splitter, and passes through the latter in order subsequently to be focused onto the detection aperture, behind which the detectors, usually photomultipliers, are situated. Detection light which does not originate directly from the focus region takes a different light path and does not pass through the detection aperture, so that point information is obtained which leads to a three-dimensional image by sequential scanning of the specimen.
A three-dimensional image is usually obtained by layer-by-layer imaging. In this case, the specimen is scanned in the axial direction (z-direction) usually by so-called specimen scanning, in which the specimen is moved in the z-direction with the aid of the specimen stage. However, this can also be achieved by displacing the objective in the axial direction, which is accompanied by a displacement of the focus of the illumination beam.
The reflected or fluorescent light emitted by the specimen when illuminated travels via a beam splitter to a detector having an entry pinhole. The power of the light coming from the specimen is measured depending on the position of the scanning beam preferably at fixed time intervals. As a result, it is possible to scan the specimen raster point by raster point in three dimensions and to determine for each scanning point a measured value which is representative of this specimen point.
With the lasers that are usually used as illumination sources in scanning microscopes, the specimen can be optimally illuminated for achieving the desired result. However, image defects always accompany the imaging of the specimen by the optical components of the microscope that are involved, which image defects may be caused by aberration, for example. Said imaging defects can be classified into geometrical and chromatic imaging defects. In monochromatic microscopy, in which the specimen is illuminated by the light of a single wavelength, in order to observe the light reflected from the specimen, only the geometrical imaging defects have to be taken into account. In fluorescence microscopy or when using a polychromatic illumination source, the chromatic imaging defects must additionally be taken into account. The image defects can in part be avoided by using high-quality, corrected optical elements. However, such high-quality elements, in particular complex corrected microscope objectives, are very expensive. Moreover, such corrected optical elements usually comprise more optical sub elements than optical elements which are corrected only slightly or not at all; thus, apochromatic microscope objectives usually comprise 10 to 15 different lenses. The multiplicity of optical sub elements means that the transmissivity of the optical system, that is to say of the optic, inevitably suffers compared with elements that are not corrected or are only slightly corrected.
It is an object of the present invention, therefore, to propose a simple and cost-effective method for compensating for imaging defects.
This object is achieved by a method for compensating for imaging defects in scanning microscopy comprising the steps of:
scanning a specimen by a light beam at raster points, which define a scanning line, whereby the light beam is guided by a scanning device through a microscope optic,
determining an imaging defect of the microscope optic,
determining a correction value for at least one raster point from the imaging defect,
calculating a corrected scanning line with respect to the correction value,
scanning the specimen at raster points defined by the corrected scanning line.
It is another object of the invention to disclose a scanning microscope with improved image quality.
This object is achieved by a scanning microscope comprising:
a light source for emitting a light beam,
a microscope optic for focusing the light beam onto a specimen,
a scanning device for scanning the specimen by the light beam at raster points, which define a scanning line,
means for determining the imaging defect of the microscope optic,
means for determining correction values for at least one raster point,
means for calculating a corrected scanning line from the correction values.
The invention has recognized that imaging defects are compensated for by suitably influencing the control of the impinging of the scanning radiation on the specimen. In particular, a beam deflection device provided for influencing the point at which the scanning radiation impinges can be influenced in accordance with the correction values determined. In the case of specimen scanning, however, it is also possible for the control of the position of the specimen stage on which the specimen to be examined is applied to be influenced in this way. As an alternative or in addition to this, it is also possible to influence the scanning speed in accordance with the correction values. This means that the imaging defects that are always inherently present in optical systems or optics can already be compensated for during the image recording. In this case, the correction value determined can be used in such a way that the scanning speed and/or the scanning path are influenced in such a way that the imaging defects of the optics are compensated for.
In order to determine the imaging defect, preferably the optic used is firstly measured. For this purpose, it is possible for a specimen that is known sufficiently precisely to be scanned in a reference measurement. In a further method step, the image of the specimen is measured and examined for image defects. A special algorithm, which can be realized e.g. in the form of an image processing program, can then determine a correction value for each raster point. By way of example, for this purpose, reference image data can be compared with the image data actually determined and a correction value can thus be determined for each raster point. This correction value thus determined is converted into a control correction signal in a further step and then taken into account in the scanning of a specimen.
As described, the correction value can be determined either in one step or iteratively. An iterative procedure may also be based on beginning with randomly determined correction values in order gradually to bring about an optimization in further iteration steps. A genetic algorithm is preferably used for this.
A scanning microscope according to the invention, which has a scanning beam which is directed via an optic onto a specimen in order to scan the latter, has means for determining the imagining defects of the optic used, means for determining correction values for at least one raster point and means for influencing the impinging of the scanning beam on the specimen.
In this case, a computer with image processing software is preferably used for determining the imaging defect of the optic. The correction values which have been determined in accordance with the specified method are advantageously stored in a memory for all the objectives used, in particular for the objectives kept in an objective turret. The correction values are transferred to the control unit, in which they are converted into control signals which drive the beam deflection device. In a concrete embodiment, e.g. the current driving the galvanometer can be correspondingly controlled by the correction values.
However, the correction values can also be transferred to a control unit in which they are converted into control signals for the specimen stage control and are used for controlling the movement of the specimen stage and thus indirectly for controlling the position of the focus in the specimen. In this case, by way of example, in order to compensate for the image field curvature, the focus of the illuminating light beam is displaced in the axial direction relative to the specimen during the image recording. For this purpose, use may be made either of a driven specimen stage or of an objective which can be displaced by a motor, in which case the driving motor, which may be e.g. an electric motor, a stepper motor or an ultrasonic motor, is addressed with the corresponding control correction signals.
In the case of sequential illumination with light of varying wavelength, a longitudinal chromatic aberration of the scanning microscope optic can be compensated for by suitable driving of the axial displacement apparatus of the focus. Analogously, suitable beam deflection device control according to the invention enables a chromatic difference of magnification to be compensated for.
Since principally geometrical imaging defects can be corrected with the aid of the method according to the invention, the scanning microscope according to the invention preferably comprises optical elements which have largely been chromatically corrected in the known manner, in which case the compensation can be performed according to the invention essentially without adversely affecting the costs of the optical imaging device.